Recombinant Aequorin

An improved method of producing recombinant aequorin was devised based on the fact that the expression of the peak amount of apoaequorin in bacterial cells occurs several hours before the secretion into culture medium (Shimomura and Inouye, 1999). The cells containing apoaequorin in the periplasmic space, before secretion, are extracted under a very mild condition and, at the same time, converted into aequorin. The purification of the extract by two steps of column chromatography yields a high-purity preparation of recombinant aequorin. 

X-ray structure of aequorin (Head et al., 2000). X-ray crystallography was performed with the recombinant aequorin prepared by the improved method described above. The crystals of recombinant aequorin were grown in a high concentration of ammonium sulfate. The results revealed that aequorin is a globular molecule containing a... 

Table 4.1.4 Semisynthetic Aequorins derived from Recombinant Aequorin (Shimomura et al., 1993a)... 

Fig. 4.1.14 Relationship between Ca2+ concentration and the initial light intensity of various recombinant semisynthetic aequorins and w-aequorin J (a semisynthetic natural aequorin made from isoform J). The curve number corresponds to the number of semisynthetic aequorin used in Table 4.1.4. A sample aequorin (3 (Ag) was in 3 ml of calcium-buffer solution containing 1 mM total EGTA, 100 mM KC1,1 mM Mg2+ and 1 mM MOPS (pH 7.0), at 23-24°C. From Shimomura etal., 1993a, with permission from Elsevier.

Fig. 4.1.15 Comparison of the luminescence and fluorescence emission spectra of natural aequorin (left panel) and recombinant e-aequorin (right panel) the luminescence spectra of Ca2+ -triggered reaction (dark solid lines), the fluorescence emission spectra of the spent solution containing 2 mM Ca2+ (dashed lines), and the luminescence spectra of the spent solution after addition of coelenterazine (light solid lines). Reproduced with permission, from Shimomura, 1995d. the Biochemical Society.

Preparation of semisynthetic aequorins. The best yield of semisynthetic aequorins can be obtained by using the apoaequorin prepared from aequorin immediately before use. Apoaequorin stored, even for 2-3 days, or recombinant apoaequorin isolated from a bacterial culture will give a significantly lower yield due to their somewhat unfolded molecular conformation. Fresh apoaequorin prepared by the Ca2+-triggered luminescence reaction appears to have the conformation best suited for regeneration. 

A cDNA encoding apoobelin was obtained from O. longissima and sequenced (Illarionov et al., 1995). The deduced amino acid sequence of the apoobelin consists of 195 amino acid residues, with a calculated molecular mass of about 22.2 kDa, closely matching the apoproteins of other Ca2+-sensitive photoproteins such as aequorin from the jellyfish Aequorea (Inouye et al., 1985 Prasher et al., 1985) and clytin from the jellyfish Phialidium gregarium (Inouye and Tsuji, 1993). To obtain recombinant apoobelin, the cDNA encoding apoobelin was expressed in E. coli (Illarionov et al., 2000). The recombinant apoobelin produced was purified and converted into obelin by incubation with coelenterazine in the presence of molecular oxygen and 2-mercaptoethanol or dithioerythritol, as in the case of aequorin. 

Knight, M. R., Read, N. D., Campbell, A. K., and Trewavas, A. J. (1993). Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins. J. Cell Biol. 121 83-90. 

Masuda, H., et al. (2003). Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin. Protein Expression and Purification 31 181-187. 

Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992). Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature 358 325-327. 

Sakaki, Y., et al. (1988). Structure and function of the calcium-binding photoprotein aequorin studies by recombinant DNA technology. In Yagi, Y., and Miyazaki, T. (eds.), Calcium Signal Cell Response, pp. 151-156. Jpn. Sci. Soc. Press Tokyo, Japan. 

Shimomura, O., and Inouye, S. (1996). Titration of recombinant aequorin with calcium chloride. Biocbem. Biophys. Res. Commun. 221 77-81. 

Shimomura, O., Inouye, S., Musicki, B., and Kishi, Y. (1990). Recombinant aequorin and recombinant semi-synthetic aequorins. Biochem. J. 270 309-312. 

Stults, N. L., et al. (1992). Use of recombinant biotinylated aequorin in microtiter and membrane-based assays Purification of recombinant aequorin from Escherichia coli. Biochemistry 31 1433-1442. 

Stults, N. L., Rivera, H. N., Burke-Payne, J., and Smith, D. F. (1997). Preparation of stable covalent conjugates of recombinant aequorin with proteins and nucleic acids. In Hastings, J. W., et al. (eds.), Biolumin. Chemilumin., Proc. Int. Symp., 9th, 1996, pp. 423-426. Wiley, Chichester, UK. 

Obelin is a Ca2+-activated bioluminescent photoprotein that has been isolated from the marine polyp Obelia longissima. Binding of calcium ions determines a luminescent emission. The protein consists of 195 amino acid residues [264] and is composed of apoobelin, coelenterazine, and oxygen. As aequorin, it contains three EF-hand Ca2+-binding sites and the luminescent reaction may be the result of coelenterazine oxidation by way of an intramolecular reaction that produces coelenteramide, C02, and blue light. As for aequorin, the luminescent reaction of obelin is sensitive to calcium and the protein was used in the past as an intracellular Ca2+ indicator. More recently, the cloning of cDNA for apoobelin led to the use of recombinant obelin as a label in different analytical systems. 

A Ca +-binding protein isolated from jellyfish Aequorea sp.) that is frequently used as a chemiluminescent calcium indicator.Aequorin contains a hydrophobic prosthetic group, coelenterazine. Investigators have been able to express the transfected gene recombinantly in a number of cells, and upon addition of the cofactor, one can measure intracellular calcium concentration. See Fura-2... 

R. Rizzuto, A. W. M. Simpson, M. Brini. and T. Pozzan. Rapid Changes of Mitochondrial Ca2+ Revealed by Specifically Targeted Recombinant Aequorin, Nature 1992, 358, 325 A. Toda, P. Pasini, M. Guardigli, M. Baraldini,... 

Zatta PE, Nyame K, Cormier Ml, Mattox SA, Prieto PA, Smith DF, Cummings RD. A solid-phase assay for beta-1,4-gaIactosyI-transferase activity in human serum using recombinant aequorin. Anal. Biochem. 1991 194 185-191. 

Immunometric assays for TSH are available commercially as manual kit procedures or for use on automated systems. For practical reasons, nonisotopic methods dominate the market and have replaced radioactive tracer methods in most routine laboratories. The majority of immunometric TSH assays label the detection antibody with chemiluminescent labelled molecules other labels include peroxidase or alkaline phosphatase and sensitive photo-metric and fluorescenri molecules. Other assays are based on the use of fluorescent labels using europium chelates chemiluminescent compounds such as acri-dinium esters or ruthenium or bioluminescent molecules such as recombinant aequorin. ... 

The purification of aequorin from jellyfish is laborious and has a low yield - two tons of jellyfish are needed to obtain about 125 mg of protein [168]. Today, aequorin can be produced by recombinantly expressing apoaequorin in E. coli followed by in vitro reconstitution with coelenterazine [13]. 

In 1975, we demonstrated that treating spent aequorin with coelenterazine in the presence of oxygen results in the regeneration of original aequorin, consequently proving that aequorin contains a coelenterazine moiety. It was an important discovery that provided the basis for producing recombinant aequorin from apoaequorin. In 1978, we proposed that aequorin contains coelenterazine-2-hydroperoxide, based on the chemical properties and reactions of aequorin. The peroxide structure was confirmed by C-NMR spectrometry in 1986."... 

Shimomura O, Musicki B, BCishi Y. Semi-synthetic aequorin an improved tool for the measurement of calcium ion concentration. Biochem. J. 1988 251, 405-410 Shimomura O, Musicki B, Kishi Y. Semi-synthetic aequorins with improved sensitivity to Ca " ions. Biochem. J. 1989 261 913-20 Shimomura O, Musicki B, Kishi Y, Inouye S. Light-emitting properties of recombinant semi-synthetic... 

Shimomura O, Inouye S. The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin. Protein Expres. Purif. 1999 16 99-5 Head JF, Inouye S, Teranishi K, Shimomura O. The crystal stmcture of the photoprotein aequorin at 2.3A resolution. Nature 2000 405 372-6. 

Brini M, Pinton P, Pozzan T, Rizzuto R. Targeted recombinant aequorins tools for monitoring Ca " in the various compartments of a living cell. Microsc Res Technique 1999 46 380-9. 

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